Protective effects of macrophage-specific integrin α5 in myocardial infarction are associated with accentuated angiogenesis

Macrophages sense changes in the extracellular matrix environment through the integrins and play a central role in regulation of the reparative response after myocardial infarction. Here we show that macrophage integrin α5 protects the infarcted heart from adverse remodeling and that the protective actions are associated with acquisition of an angiogenic macrophage phenotype. We demonstrate that myeloid cell- and macrophage-specific integrin α5 knockout mice have accentuated adverse post-infarction remodeling, accompanied by reduced angiogenesis in the infarct and border zone. Single cell RNA-sequencing identifies an angiogenic infarct macrophage population with high Itga5 expression. The angiogenic effects of integrin α5 in macrophages involve upregulation of Vascular Endothelial Growth Factor A. RNA-sequencing of the macrophage transcriptome in vivo and in vitro followed by bioinformatic analysis identifies several intracellular kinases as potential downstream targets of integrin α5. Neutralization assays demonstrate that the angiogenic actions of integrin α5-stimulated macrophages involve activation of Focal Adhesion Kinase and Phosphoinositide 3 Kinase cascades.

Editorial Note: Parts of this Peer Review File have been redacted as indicated to remove third-party material where no permission to publish could be obtained.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): This manuscript by Li et al. comes from a well established laboratory and deals with a timely and interesting topic, the role of macrophages in post-MI healing.The tested hypotheses are relevant and novel, and the provided data support the drawn conclusions.The work is technically sound.The authors describe that the integrin receptor a5, expressed by macrophages, has a role in infarct healing and post-MI recovery.Using two different cre drivers, the receptor was deleted in macrophages and interesting phenotypes reported.The selection of the deleted receptor was guided by initial transription screening.The used Cre lines make sense, and their agreement is encouraging.KO led to larger ventricles over time, indicating worse infarct healing (this information, shown in 3D and 4B should be added to the bastract, which may in general profit from some editing as it is fairly confusing, with some seemingly irrelevant information on methodology that could be cut).Mechanistically, the impared healing is due to reduced Vegfa production by macrophages, which compromises angiogenesis and recovery.There is also data on the intracellular signaling that leads to the observed phenotypes.Overall, this is a fairly complete, data rich paper that deals with an important topic.I don't see any major shortcomings.
Given the amount of data already in this manuscript, I am hesitant to request more experiments.One suggestion however would be to study the number of macrophages and their blood progenitors (monocytes) by FACS.The authors could also put this as a suggestion into the discussion.

Reviewer #2 (Remarks to the Author):
In the manuscript, "Macrophage-specific a5 integrin protects the infarcted heart from adverse remodeling, by stimulating angiogenesis through PI-3K and FAK pathways," Li et al.
demonstrate the integral role of integrin a5 in macrophages in response to cardiac injury.
The authors show that integrin a5 is upregulated on the 7th day following myocardial infarction and that upregulation is partly due to the expression of integrin a5 on the infiltrating macrophages.The authors use two different mouse models to demonstrate that macrophage-specific integrin a5 regulates vascularization of the infarct zone by regulating the expression of an important angiogenic factor, VEGFA.The authors show that PI3K and FAK signaling downstream of integrin a5 regulate VEGFA transcription.Overall, this is a nice and well-done study that will greatly interest the public.
Major Comments: 1) Can the authors show whether or not VEGF A protein is secreted by macrophages upon stimulation of integrin a5 (e.g.perform ELISA for VEGFA following integrin a5 stimulation by Fn1 or any other way)?
2) Can authors exclude the role of Cre expression in their phenotype?There are no Cre+ control mice in their study.In this study the authors report on Integrin alpha5 (ITGA5) as one of the most induced integrins in cardiac macrophages shortly after myocardial infarction in mice.
Mice with myeloid cell-specific genetic deletion (LyzM-Cre) of Itga5 show modest effects on cardiac function and infarct size, and reduced angiogenesis at later time points (28d) upon infarction.The authors report a similar phenotype in a similar mouse model, where Cx3cr1-Cre is used to delete Itga5 in macrophages.Mechanistically, the authors report that either genetic deletion of Itga5 in macrophages or application of an antibody to the latter suppresses activation of the PI3kinase and FAK-pathway and VEGFA mRNA and protein.To this reviewer the study contains a number major limitations, that do not justify the major claims posed by this study.

Major points:
1) The authors should define more clearly what cells express IGTA5 and whether the cells sequenced in Fig. 1 are indeed pure macrophages.Cd11b+/Ly6G-magnetic bead selection is used to purify cardiac macrophages.As this one step procedure often leads to contaminations/noise from other cell types, was the cell fraction controlled for impurities?Flow cytometry measurement should be able to dissect the level of EC (CD31), FB (collagens, Pdgfrb) or cardiomyocyte contamination.This reviewer could not find data for the purity of the cell fractions studied.This point appears of considerable relevance, as the composition of the myeloid cell fraction drastically changes upon myocardial infarction.So perhaps the 'upregulation' of ITGA5 may turn out also result from different cell populations infiltrating the myocardium.
2) For their RNA-Seq experiment in Figure 1, they use CD11b as a macrophage marker, for immunofluorescence staining, however, they use CSF1R, yet this receptor is also expressed on monocytes?If the authors want to make the distinction, a macrophage-specific gene/protein such as Cd68 or F4/80 should be stained as well.
3) For analysis of infiltration parameters, Mac2/Galectin3 was used to stain the macrophage population.While Galectin3 is an important molecule for migration and infiltration, it also might stain/identify monocytes.Why was this staining chosen instead of CSF1R or a more Mac-specific protein?
4) The experiments in figure 6  My main concern with this paper is that the ITGA5 expressing "macrophage" cell population(s) remain poorly defined (e.g.resident vs. recruited).Defining macrophages as CD11b+/Ly6G-(Figure 1) is too simplistic.Immunostaining images presented in Figure 2 are poor quality.FACS should be used to better define monocyte/macrophage subsets expressing ITGA5 during AMI healing (time course).Considering that ITGA5 is a cell surface protein it might be included in more sophisticated FACS panels.Moreover single (myeloid) cell RNAseq should be used to better define ITGA5-expressing myeloid cell subsets.
Similar considerations apply when studying ITGA5 effects on post AMI inflammation (Mac2 immunofluorescence staining alone is not sufficient; Suppl.Figure III).The authors should use established flow cytometry panels to assess the impact of macrophage-specific ITGA5 deletion on the inflammatory response after AMI (studying diverse myeloid and lymphoid cell subsets).
Considering that the majority of patients with AMI undergoes reperfusion therapy, the impact of LyzM-Cre deletion on LV remodeling and angiogenesis should be briefly explored/confirmed in a transient coronary artery ligation mouse model.Is a ca.20% reduction in VEGF expression in ITGA5 KO infarct macrophages (detected by angiogenesis PCR array in Figure 7A) really sufficient to explain the more adverse left ventricular remodeling after AMI in the KO mice?How does the ITGA5 KO impact overall left ventricular VEGF expression levels?Are non-angiogenesis related growth factors/genes affected by macrophage ITGA5 KO?For example, genes that have a direct impact on scar maturation (Figure 4)?

Minor:
Line 53: authors should not speculate about ischemic cardiomyopathy as this is not examined in this manuscript.
Line 295: grammar? Figure 1A: is each line representing 1 mouse?Suppl.Figure XI: there appears to be a large interindividual variability within the WT and KO groups (3 each).This requires a comment.

RESPONSE TO THE REVIEWERS:
We are grateful to the reviewers for their comments that improved the quality of our work.We have performed extensive new experiments to address the reviewers' criticisms: 1.In order to better define the macrophage identity of ITGA5+ cells, we have performed new flow cytometry experiments that clearly demonstrate increased ITGA5 levels in infarct macrophages, 7 days after myocardial infarction.The new data are shown in Figure 1A-C (the gating strategy is illustrated in Supplemental Figure II).
New flow cytometry experiments were also performed to examine the effects of myeloid cellspecific ITGA5 loss on infiltration of the infarct with leukocytes.The new data are shown in Figure 4, and the gating strategy is presented in Supplemental Figure VI.The findings are consistent with the immunofluorescence studies, suggesting no significant effects of myeloid cell-specific ITGA5 loss on myeloid cell, macrophage and lymphocyte infiltration.
2. New single cell RNAseq experiments were performed to better define the macrophage clusters that express various integrins and to identify relations between integrin expression and specific phenotypes.We have performed scRNA-seq on cells sorted from control and infarcted CSF1R EGFP macrophage reporter mice.The new data are shown in Figures 7 and 8, in Supplemental Figures XVII and XVIII, and in Supplemental Tables I-III The findings show cluster-specific patterns of integrin expression in infarct macrophages.Importantly, the cluster exhibiting high expression of Itga5, has high expression levels of several angiogenic genes, including Vegfa, Vegfb, Angpt2, Angptl2, Angptl4 and Angptl6.The high Itga5 expression in the angiogenic cluster of macrophages further supports our myeloid cell-specific and macrophage-specific in vivo studies on the role of ITGA5 in stimulation of an angiogenic macrophage program.
3. We have explored additional mediators that may be responsible for the effects of myeloid cell and macrophage-specific ITGA5 loss.We used our RNAseq analysis of the profile of infarct macrophages to identify mediators with roles in inflammation, repair and matrix remodeling, which are differentially regulated by ITGA5 loss.Because many of these genes may reflect secondary consequences of the effects on remodeling (rather than primary targets of ITGA5 in macrophages), we examined which of these genes are modulated in a similar manner by ITGA5 blockade in vitro.The data are presented in Supplemental Table XI and presented in the results section.We found that expression of emilin2, encoding the angiogenic matricellular protein EMILIN2 (elastin microfibril interface located protein 2), and Ecm1 (which encodes the matrix protein Extracellular matrix protein 1 (ECM1), a potent stimulus of endothelial cell proliferation) was consistently suppressed upon ITGA5 disruption in vivo and in vitro.Thus, ECM1 and EMILIN2 may be additional ITGA5-dependent angiogenic mediators secreted by infarct macrophages.

4.
We have addressed all other concerns raised by the reviewers.We present new immunofluorescence images to clearly show the ITGA5+ macrophages (Figure 1D show new data on VEGFA protein levels (Figure 9J).We have also clarified important points made by the reviewers.
Overall, the revised version contains several new figures (Figures 1, 4, 7, 8, Supplemental Figures II, III, VI, X, XIII, XVII, XVIII) and Tables (Supplemental Tables I, II, III, XI, XII, XIII).Drs Yang Liu, Richard Ma, Izabela Tuleta, Harikrishnan Venugopal, Fenglan Zhu, Kai Su, Jinghang Zhang and Deyou Zheng who contributed to the new scRNA-seq, flow cytometry, and immunofluorescence experiments presented in the revised version were included as co-authors.We have addressed the specific concerns raised by the reviewers as follows:

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): This manuscript by Li et al. comes from a well established laboratory and deals with a timely and interesting topic, the role of macrophages in post-MI healing.The tested hypotheses are relevant and novel, and the provided data support the drawn conclusions.The work is technically sound.The authors describe that the integrin receptor a5, expressed by macrophages, has a role in infarct healing and post-MI recovery.Using two different cre drivers, the receptor was deleted in macrophages and interesting phenotypes reported.The selection of the deleted receptor was guided by initial transcription screening.The used Cre lines make sense, and their agreement is encouraging.KO led to larger ventricles over time, indicating worse infarct healing (this information, shown in 3D and 4B should be added to the abstract, which may in general profit from some editing as it is fairly confusing, with some seemingly irrelevant information on methodology that could be cut).Mechanistically, the impaired healing is due to reduced Vegfa production by macrophages, which compromises angiogenesis and recovery.There is also data on the intracellular signaling that leads to the observed phenotypes.Overall, this is a fairly complete, data rich paper that deals with an important topic.I don't see any major shortcomings.
Thank you very much for your kind and insightful comments, and for your helpful recommendations.We have revised the abstract to clarify the message and also to comply to the journal's word limits.
Given the amount of data already in this manuscript, I am hesitant to request more experiments.One suggestion however would be to study the number of macrophages and their blood progenitors (monocytes) by FACS.The authors could also put this as a suggestion into the discussion.
Thank you for your comment.We have performed new flow cytometry experiments, in order to study the time course of ITGA5 expression in macrophages, and to compare infiltration of leukocytes, macrophages and lymphocytes between myeloid cell-specific ITGA5 KO and control infarcts.
Flow cytometry for ITGA5 and macrophage markers is shown in Figure 1 (the gating strategy is illustrated in Supplemental Figure II).The findings show that the number of ITGA5+ macrophages peaks 7 days after infarction.The findings are consistent with the immunofluorescent staining (Figure 1) and RNA-seq data (Supplemental Figure I), which suggests a similar time course of ITGA5 protein expression in macrophages infiltrating the infarct.
Moreover, we have performed new flow cytometry experiments to examine the effects of myeloid cell-specific ITGA5 loss on macrophage and lymphocyte infiltration.The new data are shown in Figure 4, and the gating strategy for this experiment is illustrated in Supplemental Figure VI.The findings show no significant effects of myeloid cell-specific ITGA5 loss on recruitment of myeloid cells, macrophages and T lymphocytes in the healing infarct.Thus, these observations are in agreement with the immunofluorescence data shown in Supplemental Figure VII.
Reviewer #2 (Remarks to the Author): In the manuscript, "Macrophage-specific a5 integrin protects the infarcted heart from adverse remodeling, by stimulating angiogenesis through PI-3K and FAK pathways," Li et al. demonstrate the integral role of integrin a5 in macrophages in response to cardiac injury.The authors show that integrin a5 is upregulated on the 7th day following myocardial infarction and that upregulation is partly due to the expression of integrin a5 on the infiltrating macrophages.The authors use two different mouse models to demonstrate that macrophage-specific integrin a5 regulates vascularization of the infarct zone by regulating the expression of an important angiogenic factor, VEGFA.The authors show that PI3K and FAK signaling downstream of integrin a5 regulate VEGFA transcription.Overall, this is a nice and well-done study that will greatly interest the public.
Thank you very much for your kind and insightful comments, and for your helpful recommendations.
Major Comments: 1) Can the authors show whether or not VEGF A protein is secreted by macrophages upon stimulation of integrin a5 (e.g.perform ELISA for VEGFA following integrin a5 stimulation by Fn1 or any other way)?
We have performed new experiments examining the effects of ITGA5 neutralization on VEGFA protein levels.Consistent with the mRNA data, the protein analysis shows that VEGFA secretion by isolated macrophages, cultured in the presence of fibronectin, is dependent on ITGA5.The new data are shown in Figure 9J.
2) Can authors exclude the role of Cre expression in their phenotype?There are no Cre+ control mice in their study.
Thank you very much for raising this important point.To address this question, we have performed new experiments examining the effects of Cre (described below).However, first we would like to explain the basis for our initial use of floxed controls.There is extensive evidence that Cre expression has effects on cardiomyocyte function; for this reason, Cre controls are routinely included in most investigations examining cardiomyocyte-specific manipulations, especially when the MCM system is used.In contrast, in investigations examining the role of macrophages through the use of the Cre-lox system, the vast majority of studies have used floxed controls, as there has been no evidence on Cre actions on macrophage phenotype.To illustrate this, we have reviewed manuscripts examining macrophage-specific actions using the LysM Cre and CX3CR1 CreER drivers that were recently (2019-2022) published in Nature Communications and in other high-impact journals.Virtually all the studies we identified (Reviewer Table I and II) used floxed controls, ignoring any possible actions of Cre recombinase.The absence of significant Cre effects is also supported by our own published work comparing effects of Smad2 and Smad3 deletion in myeloid cells using the LysM Cre driver.Our studies showed that although myeloid cell-specific Smad3 loss perturbed phagocytosis and impaired suppression of post-infarction inflammation 1 , myeloid cell-specific Smad2 deletion had no significant effects 2 .The absence of significant differences between LysM Cre ;Smad2fl/fl and Smad2 fl/fl animals following infarction 2 suggests that Cre expression in myeloid cells has no major impact on their phenotype.
Reviewer Table I However, as the reviewer points out, Cre effects should be considered in all genetic manipulation studies.Thus, in order to exclude that the observed effects are due to Cre expression (rather than ITGA5 loss) we have performed new experiments examining the effects of Cre on the main positive endpoints of our study: adverse remodeling and infarct angiogenesis in both the LysMCre and CX3CR1CreER models.LysMCre/+ animals and Cre-/-controls underwent infarction protocols and were sacrificed 7 days after coronary occlusion (the timepoint in which myeloid cellspecific ITGA5 KO mice had worse remodeling and reduced microvascular density).Echocardiography showed that Cre expression did not affect systolic function and dilative remodeling.Moreover, microvascular density , assessed through CD31 immunohistochemistry was comparable between Cre+ and Cre-negative animals.The data are shown in Supplemental Figure X.
Similarly, we performed new experiments using tamoxifen-treated CX3CR1CreER mice and Crenegative controls.There were no significant differences in echocardiographic parameters, in microvascular density and in the density of mature coated vessels between groups.The data are shown in Supplemental Figure XIII.No adjustment for multiple comparisons was used for the array data presented in this figure.This is now clearly indicated in the figure legend.In this study the authors report on Integrin alpha5 (ITGA5) as one of the most induced integrins in cardiac macrophages shortly after myocardial infarction in mice.
Mice with myeloid cell-specific genetic deletion (LyzM-Cre) of Itga5 show modest effects on cardiac function and infarct size, and reduced angiogenesis at later time points (28d) upon infarction.The authors report a similar phenotype in a similar mouse model, where Cx3cr1-Cre is used to delete Itga5 in macrophages.Mechanistically, the authors report that either genetic deletion of Itga5 in macrophages or application of an antibody to the latter suppresses activation of the PI3kinase and FAK-pathway and VEGFA mRNA and protein.To this reviewer the study contains a number major limitations, that do not justify the major claims posed by this study.
Thank you very much for the insightful comments and criticisms.We have extensively revised our study following your recommendations.
Major points: 1) The authors should define more clearly what cells express IGTA5 and whether the cells sequenced in Fig. 1 are indeed pure macrophages.Cd11b+/Ly6G-magnetic bead selection is used to purify cardiac macrophages.As this one step procedure often leads to contaminations/noise from other cell types, was the cell fraction controlled for impurities?Flow cytometry measurement should be able to dissect the level of EC (CD31), FB (collagens, Pdgfrb) or cardiomyocyte contamination.This reviewer could not find data for the purity of the cell fractions studied.This point appears of considerable relevance, as the composition of the myeloid cell fraction drastically changes upon myocardial infarction.So perhaps the 'upregulation' of ITGA5 may turn out also result from different cell populations infiltrating the myocardium.
2) For their RNA-Seq experiment in Figure 1, they use CD11b as a macrophage marker, for immunofluorescence staining, however, they use CSF1R, yet this receptor is also expressed on monocytes?If the authors want to make the distinction, a macrophage-specific gene/protein such as Cd68 or F4/80 should be stained as well.
We agree with the reviewer that immunomagnetic sorting can be associated with contamination with other cell types.Thus, although the vast majority of the cells used for RNA-seq are macrophages, we cannot exclude contamination with other cell types that may have influenced the results.For this reason, we have performed extensive new experiments to examine the time course of ITGA5 expression in infarct macrophages.
a. We have examined the time course of ITGA5 expression in macrophages using flow cytometry.
The findings support the immunohistochemical data, demonstrating that expression of ITGA5 in CD11b+/Ly6G-/CD64+/MerTK+ macrophages peaks 7 days after MI.The new data are shown in Figure 1.The gating strategy for this experiment is shown in Supplemental Figure II.
b.We have also performed new scRNAseq data examining the transcriptomic profile of sorted CSF1R+ cells harvested from sham and infarcted hearts.We identified 12 clusters of CSF1R+ cells.The data are presented in figures 7 and 8, and in Supplemental Figures XVII and XVIII and in Supplemental Tables I, II and III.Our findings showed cluster-specific patterns of Itga5 expression in infarct macrophages.Interestingly, a cluster with high expression of angiogenic genes, including Vegfa, Vegfb, Angpt2, Angptl2, Angptl4 and Angptl6 was identified, representing ~5% of the CSF1R+ cells in the infarct.These cells exhibited higher Itga5 expression levels than other clusters (Figure 8, Supplemental Table III).These findings support our in vivo targeting data, which suggest a role for macrophage ITGA5 in mediating an angiogenic macrophage phenotype.
3) For analysis of infiltration parameters, Mac2/Galectin3 was used to stain the macrophage population.While Galectin3 is an important molecule for migration and infiltration, it also might stain/identify monocytes.Why was this staining chosen instead of CSF1R or a more Mac-specific protein?
We have previously performed a systematic study to identify the optimal macrophage marker for staining of formalin-fixed paraffin embedded sections, using 2 different reporter lines (CSF1REGFP and CX3CR1EGFP mice) and several different antibodies 17 .Of the antibodies we tested, Mac2 had the highest reproducibility for paraffin-embedded samples with complete absence of artifacts (supported by the use of Gals3 KO samples) and excellent quality of staining.
In contrast, other markers (such as F4/80 antibodies) had poor performance in paraffin-embedded tissues.Although Mac2 is not the most specific of the anti-macrophage antibodies, the reproducibility of staining (when paraffin-embedded samples are studied) make it highly suitable for quantitative analysis.
In order to strengthen our analysis of the effects of myeloid cell-specific ITGA5 loss on macrophage recruitment, we have performed new experiments using flow cytometry for assessment of macrophage infiltration in myeloid cell-specific ITGAA5 KO and control infarcts.
The new data are shown in Figure 4 (the gating strategy is illustrated in Supplemental Figure VI.The flow cytometry data support the immunofluorescence experiments, as no significant effects of myeloid cell-specific ITGA5 loss on recruitment of myeloid cells, macrophages and T lymphocytes were noted.We initiated breeding with the 2 different Cre drivers concurrently; however, as myeloid cellspecific KO mice became available first, we performed extensive work with this line.The 2 Cre drivers provide complementary information.The LyzM Cre driver is a robust tool for dissection of myeloid cell-specific effects, but lacks specificity for macrophages.The inducible CX3CR1CreER is more specific for macrophages, but may be less sensitive in targeting the broad range of macrophages infiltrating the infarct.We feel that the 2 lines provide complementary information.Moreover, the consistent results obtained with the 2 different drivers increase the level of confidence on the observed angiogenic effect of macrophage ITGA5.

5) Capillary rarefaction has been reported during cardiac remodeling in many studies.
Whether the observed (modest) effects on remodelling are causally related to macrophagemediated effects on angiogenesis appears not clear and the authors should tone down the respective claims.
We agree with the reviewer that it is unclear whether the observed perturbation in angiogenesis and in vascular maturation are responsible for the effects on adverse remodeling.However, we have performed a systematic study of alternative possibilities.Scar size at 7 days was not affected, suggesting no significant effects on cardiomyocyte loss.Myofibroblast density and collagen content were comparable between groups.No significant effects on macrophage and lymphocyte recruitment were noted by flow cytometry and immunofluorescence.Microvascular density and vascular maturation are consistently perturbed upon ITGA5 loss in myeloid cells and macrophages.A ~30% reduction in microvascular density may have a significant impact on perfusion, repair and function after myocardial infarction.To address the reviewer's concern, we have revised the discussion to indicate the limitations.
6) Figure 5: CD31 immunohistochemistry and alphaSMA immunofluorescence are used to show a reduction of angiogenesis after ITGA5 depletion in myeloid cells.For analysis, two non-adjacent sections per mouse were used.As vessels are difficult to quantify with sections, it is unclear, if two sections yield enough statistical power.Please comment.
For assessment of microvascular density and vascular maturation, we assessed more than 10 fields (200X magnification) from 2 different levels for each mouse.The high quality of the staining (which as illustrated in the figures is free of artifacts and provides highly reproducible results), the large number of fields (>20/mouse) and the large number of animals studied (ITGA5 fl/fl n=19, Myα5KO n=12, iMaα5KO n=5; 28-day group: ITGA5 fl/fl n=22, Myα5KO n=13, iMaα5KO n=9) increase the power of the quantitative analysis, which is sufficient to detect a 30% difference between groups,

7)
The in vitro experiments would benefit from a control group without Itga5 activation, i.e. in the absence of fibronectin.
In our early studies (and in our RNA-seq experiments) we studied macrophages in the presence or absence of fibronectin.Supplemental Figures XXII and XXIII show these findings, illustrating comparisons both in the presence and absence of fibronectin.However, the effects of ITGA5 neutralization were similar in the presence or absence of fibronectin.This may reflect, at least in part, the production of endogenous fibronectin by the cells.Thus, the neutralization experiments examining the mechanisms for ITGA5 actions were performed in fibronectin-treated cells.
8) The authors may comment, why they chose to study ITGA5 and not other molecules upregulated to a similar or even greater extent (ITGB5)?
Li and colleagues have explored the role of alpha5 integrin (ITGA5) expressed by macrophages during infarct healing.They have used two Cre deleter lines (constitutive LyzM-Cre and tamoxifen-inducible CX3CR1-CreER) to delete ITGA5 specifically in macrophages and found that macrophage ITGA5 promotes angiogenesis and attenuates adverse left ventricular remodeling (dilation) after acute myocardial infarction (AMI).Mechanistically, they propose that ITGA5 augments VEGF expression in macrophages via activation of PI3K and FAK.
My main concern with this paper is that the ITGA5 expressing "macrophage" cell population(s) remain poorly defined (e.g.resident vs. recruited).Defining macrophages as CD11b+/Ly6G-(Figure 1) is too simplistic.Immunostaining images presented in Figure 2 are poor quality.FACS should be used to better define monocyte/macrophage subsets expressing ITGA5 during AMI healing (time course).Considering that ITGA5 is a cell surface protein it might be included in more sophisticated FACS panels.Moreover single (myeloid) cell RNAseq should be used to better define ITGA5-expressing myeloid cell subsets.
Thank you very much for the insightful comments and criticisms.We have extensively revised our study following your recommendations.
We have performed both flow cytometry and single cell RNA-seq experiments to address the concerns regarding the macrophage identity of the ITGA5+ cells: b.We have performed new scRNAseq experiments, examining the transcriptomic profile of sorted CSF1R+ cells harvested from control and infarcted hearts (7 days after coronary occlusion).We identified 12 clusters of CSF1R+ cells.The data are presented in the results section and in figures 7 and 8, Supplemental Figures XVII and XVIII, and in Supplemental Tables I, II and III.The findings suggest cluster-specific patterns of integrin expression in infarct macrophages.Supplemental Table III shows all integrin genes that were specifically upregulated in one or more of the CSF1R+ cell clusters (in comparison to all other clusters).Itga5 expression was significantly higher in a cluster of macrophages that exhibited an angiogenic transcriptional profile (Angiogenic macrophages, Amp, Fig 8B ), when compared with cells from all other clusters (padj=0, logFC=0.87).The angiogenic macrophages significantly expanded after infarction, and were characterized by high expression of angiogenic genes, including Vegfa, Vegfb, Angpt2, Angptl2, Angptl4 and Angptl6.
Moreover, comparison of integrin expression levels between infarcted and control hearts showed significant Itga5 upregulation in a large cluster of macrophages that exhibited high expression of growth factors and matricellular genes (reparative macrophages, RMp) The partner chain for ITGA5, ITGB1 was broadly expressed by the majority of cells in all clusters (Fig 8C ).The Itgam (encoding ITGAM/CD11b), Itgb2 and Itgb5 genes were also broadly expressed in cells from all macrophage and monocyte clusters (Figure 8B-F).Other integrins were predominantly expressed by specific clusters.Inflammatory macrophages ( a cluster with high expression of pro-inflammatory genes that -Imp) had high expression of Itga1, ItgaL and Itgb7, in comparison to other clusters.Resident cardiac macrophages (CMp) exhibited higher expression of Itga9 in comparison to other clusters.Itgax was predominantly expressed by the angiogenic macrophage cluster and by the minor resident macrophage clusters (Mp11 and Mp12), whereas Itga6 was expressed at higher levels by reparative macrophages.Itgav was expressed by significant subpopulations of cells from all clusters (Supplemental Fig XVII ).In contrast,Itgb4, Itgae, Itga2, Itga2b, Itga3, Itga7, Itga8, Itga10, and Itga11 expression was very low in all clusters (Supplemental Fig XVIII ).
Thus, the scRNA-seq data showing that angiogenic macrophages have high expression of Itga5 are consistent with the pro-angiogenic effects of ITGA5 in macrophages, suggested by the myeloid cell and macrophage-specific KO experiments.
c.We have improved the quality of the immunofluorescence images to illustrate infiltration of the infarct with ITGA5+ macrophages.New immunofluorescence images clearly show the ITGA5+ macrophages at the 7 and28-day timepoints, by including the 2 channels and the merged image (Figure 1D).Supplemental Figure III shows the complete time course.
Similar considerations apply when studying ITGA5 effects on post AMI inflammation (Mac2 immunofluorescence staining alone is not sufficient; Suppl.Figure III).The authors should use established flow cytometry panels to assess the impact of macrophage-specific ITGA5 deletion on the inflammatory response after AMI (studying diverse myeloid and lymphoid cell subsets).
We have performed new experiments using flow cytometry to quantitatively assess the effects of myeloid cell-specific ITGA5 loss on the numbers of myeloid cells, macrophages and T cells.The findings are shown in Figure 4, whereas the gating strategy is illustrated in Supplemental Figure XI.The data show that myeloid cell-specific ITGA5 loss does not affect infiltration of the infarct with myeloid cells, macrophages and T lymphocytes.These findings are consistent with the Mac2 immunofluorescence (Supplemental Figure VII) Considering that the majority of patients with AMI undergoes reperfusion therapy, the impact of LyzM-Cre deletion on LV remodeling and angiogenesis should be briefly explored/confirmed in a transient coronary artery ligation mouse model.
Because the focus of our study is on repair, the non-reperfused infarction model is optimal for testing the hypothesis, as it results in formation of a transmural scar, which is dependent on neovessel formation in order for healing to occur.In contrast, the reperfused infarction model induces a mid-myocardial infarct, in which the role of neovessels is more limited (as the coronary is reperfused after a brief ischemic interval).In addition to this, the small mid-myocardial infarcts in ischemia/reperfusion models make assessment of microvascular density more challenging, and perhaps less meaningful.Reviewer Figure I illustrates this.
We agree with the reviewer that the reperfused MI model could provide valuable information; however, this information would address a different question: whether integrin-dependent activation of macrophages has an impact on acute cardiomyocyte death following ischemia (which is not affected in the non-reperfused infarction model, as all cardiomyocytes in the area at risk die).
Reviewer Figure I: H&E section illustrating the midmyocardial localization of infarction in reperfused infarcts (60 min coronary occlusion/7 days of reperfusion).Rapid restoration of flow in the infarct zone in reperfused infarction would be expected to limit any effects of changes in angiogenesis.Moreover, the non-transmural nature of the infarct (arrows) makes assessment of infarct angiogenesis challenging.Considering that the goal of the study is to examine the role of macrophage ITGA5 signaling in cardiac repair, we felt that the non-reperfused infarction model is optimal to test the hypothesis.The reperfused MI model could provide valuable information to understand any effects of integrin macrophage signaling on acute cardiomyocyte death (which is not affected in the non-reperfused infarction model, as all cardiomyocytes in the area at risk die).
Is a ca.20% reduction in VEGF expression in ITGA5 KO infarct macrophages (detected by angiogenesis PCR array in Figure 7A) really sufficient to explain the more adverse left ventricular remodeling after AMI in the KO mice?How does the ITGA5 KO impact overall left ventricular VEGF expression levels?Are non-angiogenesis related growth factors/genes affected by macrophage ITGA5 KO?For example, genes that have a direct impact on scar maturation (Figure 4)?
These are excellent points; we greatly appreciate the recommendations.As a general rule, we feel it is unlikely that a single mediator can explain the functional consequences of deletion of a key upstream signal with broad effects on cellular phenotype.However, VEGFA is a very good candidate, due to its high level of expression, consistent changes in vivo and in vitro, and important function in a relevant cellular response (angiogenesis).Thus, we have used Vegfa as a reliable readout to dissect the mechanisms responsible for ITGA5-mediated actions in macrophages.To address the reviewers' comments, we have performed additional experiments to assess VEGFA protein levels and to identify additional ITGA5-dependent macrophage-derived mediators that may explain the in vivo phenotype.
First, we have performed new experiments examining the effects of ITGA5 neutralization on VEGFA protein levels.Consistent with the mRNA data, the protein analysis shows that VEGFA secretion by isolated macrophages, cultured in the presence of fibronectin, is dependent on ITGA5.
The new data are shown in Figure 9J.
Second, we have identified additional ITGA5-mediated reparative genes that may explain the effects of macrophage-specific ITGA5 loss, using our in vivo and in vitro RNA-seq data.
Comparison of the transcriptomic profile of myeloid cells harvested from Mya5KO vs ITGA5 fl/fl infarcts showed that a significant number of proinflammatory genes (including Il1a, Il1b, Il6, Cxcl3 and Cxcl5), anti-inflammatory genes (such as Il10 and Tgfb2), and angiogenic mediators (such as Nrg1, Hbegf, and Areg) were downregulated in infarct macrophages upon ITGA5 disruption (Supplemental Table XI).Moreover, expression of Lox and Tgm2 (encoding the matrixcrosslinking enzymes lysyl-oxidase and transglutaminase-2 respectively) was also markedly reduced in ITGA5 KO infarct macrophages.However, differential gene expression in vivo in ITGA5 KO macrophages only in part reflects direct effects of ITGA5, as expression of various genes may be affected indirectly by the consequences of cell-specific ITGA5 loss on adverse remodeling and dysfunction.In order to identify genes specifically modulated by ITGA5 signaling, we examined which of the differentially expressed genes in vivo were also modulated in vitro.We found that expression of emilin2, encoding the angiogenic matricellular protein EMILIN2 (elastin microfibril interface located protein 2) 18 , and Ecm1 (which encodes the matrix protein Extracellular matrix protein 1 (ECM1), a potent stimulus of endothelial cell proliferation 19 ) was consistently suppressed upon ITGA5 disruption in vivo and in vitro.Thus, ECM1 and EMILIN2 may be additional ITGA5-dependent angiogenic mediators secreted by infarct macrophages.The new data are presented in the results section and in Supplemental Table XI.
Minor: Line 53: authors should not speculate about ischemic cardiomyopathy as this is not examined in this manuscript.
We have deleted the sentence (as the abstract was rewritten to comply with journal restrictions).
We have corrected this statement indicating that LyzM-Cre enables myeloid cell-specific gene deletion.
We have corrected the sentence.The significant variability is typical of the in vivo model, in which gene expression in myeloid cells is affected not only by the specific manipulation, but also by any consequences of the genetic intervention on cardiac remodeling and dysfunction.An additional factor is that the heatmap illustrates z scores (in comparison to the mean expression for the same gene in all samples).For low expression genes, this results in the appearance of high variability at expression levels; however (due to the very low expression), biological significance is unclear.We have added a brief comment in the figure legend (Supplemental Figure XIX) indicating this.Moreover, for this reason, identification of candidate ITGA5-mediated signals and pathways was based on consistent findings in both in vivo and in vitro data.
We are grateful to the reviewers and editors for their comments.We have revised our manuscript following their recommendations.
In the revised version of our manuscript, we have addressed all remaining concerns.Specifically:

We have included the quantitative data on the effects of myeloid cell-specific
ITGA5 loss on neutrophil density (Figure 4) and we have revised Figure 1 to show density plots. 5.In response to comments by Reviewer 3, we have revised the title, abstract and discussion to delete statements suggesting a causative relation between and increased capillary density and attenuated remodeling, while indicating the association.6.We have also addressed concerns by Reviewer 1 regarding bands in the stacking/running gel interface present in 2 of the unedited Western blots.We explain why these bands do not affect the quantitative analysis and the conclusions of these experiments.We also point out (and show ample evidence) that similar imperfections in Western blots are commonly found in the majority of papers published in top-quality journals (including Nature family publications, such as Nature, Nat Cell Biol, Nat Commun).7. We have addressed all other comments raised by the reviewers.

Dr Shuaibo Huang who contributed to the revisions has been added to the authors'
list.

Specifically, we have addressed the reviewers' comments as follows:
Reviewer #1 (Remarks to the Author): No further comments.
Thank you very much for your careful review of our manuscript and for your kind and insightful comments.
Reviewer #2 (Remarks to the Author): The manuscript "Macrophage-specific integrin a5 protects the infarcted heart from adverse remodeling by stimulating angiogenesis through PI3K and FAK pathways" reports an interesting observation that the deletion of integrin a5 in macrophages exacerbates cardiac response to injury.The authors performed a large number of experiments and supplied additional data in response to reviewers' critics.This reviewer believes that the injury data is interesting and compelling.However, the mechanistic aspect of the paper is weak and provided data does not fully support authors' conclusions that integrin a5 functions in macrophages to regulate VEGF A expression through the activation of FAK and PI3K.
Thank you very much for your careful review of our manuscript and for your kind and insightful comments.We have revised our manuscript following your recommendations. Major: The quality of IF is insufficient to convince the reviewer that stained cells express Itga5 and/or CSFR1.Thin confocal slices are necessary to prove the localization of Itga5 protein and CSFR1GFP signal in the same cell.
We have replaced all previous images with better quality figures and we have performed As the reviewer points out, some cells exhibit cytoplasmic labeling.This likely reflects de novo synthesis of ITGA5. and  integrin chains form heterodimers in the endoplasmic reticulum after their synthesis (1).Heterodimers are glycosylated in the ER (2), then undergo additional processing in the Golgi, until delivered to the cell membrane (3).Moreover, cell surface integrins are recycled through endocytosis (4).Thus, cytoplasmic localization of ITGA5 in infarct macrophages may reflect de novo synthesis (which is followed by shuttling to the membrane), or endocytotic cycling.
2) There appears to be either a leaky expression of CSF1RGFP or too much autofluorescence making it difficult to discern a legitimate signal from the background in Sup.

Minor Comments: 1 )Figure 2 :
Expression of Itga5 in macrophages in Fig 2A is not clearly shown.Not clear what R designates in Fig. 2A.Not clear what green and yellow stainings mark in panels 7d R and 28d R.2) Supplemental Figure VI: Itga5 expression and deletion are not clearly shown in panel C. Maybe the authors can show magnified panels as well as show separate channels.The legend to this figure does not mention arrows in panel C. 3)Supplemental Figure XI: Please mention whether or not p values in the table of panel C were adjusted for multiple testing.Reviewer #3 (Remarks to the Author): Review Li et al. " Macrophage-specific alpha5 integrin protects the infarcted heart from adverse remodeling, by stimulating angiogenesis through PI-3K and FAK pathways"

Reviewer # 4 (
use another Cre-line (CX3CR1) to recapitulate the findings of the knockout using LyzM-Cre.It is unclear why the authors didn't use this model from the start as they want to analyze the role of ITGA5 in macrophages?To what extent are the other myeloid cells affected from ITGA5 loss in the first (myeloid cell) model?What is the (mature) macrophage content within the LyzM positive myeloid cell fraction in the myocardial infarction model?5) Capillary rarefaction has been reported during cardiac remodeling in many studies.Whether the observed (modest) effects on remodelling are causally related to macrophagemediated effects on angiogenesis appears not clear and the authors should tone down the respective claims.6) Figure 5: CD31 immunohistochemistry and alphaSMA immunofluorescence are used to show a reduction of angiogenesis after ITGA5 depletion in myeloid cells.For analysis, two non-adjacent sections per mouse were used.As vessels are difficult to quantify with sections, it is unclear, if two sections yield enough statistical power.Please comment.7) The in vitro experiments would benefit from a control group without Itga5 activation, i.e. in the absence of fibronectin.8) The authors may comment, why they chose to study ITGA5 and not other molecules upregulated to a similar or even greater extent (ITGB5)?Remarks to the Author): Li and colleagues have explored the role of alpha5 integrin (ITGA5) expressed by macrophages during infarct healing.They have used two Cre deleter lines (constitutive LyzM-Cre and tamoxifen-inducible CX3CR1-CreER) to delete ITGA5 specifically in macrophages and found that macrophage ITGA5 promotes angiogenesis and attenuates adverse left ventricular remodeling (dilation) after acute myocardial infarction (AMI).Mechanistically, they propose that ITGA5 augments VEGF expression in macrophages via activation of PI3K and FAK.
, Supplemental Figure III, Supplemental Figure XI).We show new data excluding Cre effects on macrophagemediated actions in our genetic models (Supplemental Figure X, Supplemental Figure XIII.We

Minor Comments: 1 )Figure 2 :
Expression of Itga5 in macrophages in Fig 2A is not clearly shown.Not clear what R designates in Fig. 2A.Not clear what green and yellow stainings mark in panels 7d R and 28d R. 2) Supplemental Figure VI: Itga5 expression and deletion are not clearly shown in panel C. Maybe the authors can show magnified panels as well as show separate channels.The legend to this figure does not mention arrows in panel C.We have improved the quality of the immunofluorescence data and we have performed new flowcytometry experiments to study the time course of ITGA5 expression in macrophages.Figure1in the revised version of the manuscript shows new flow cytometry data demonstrating an increased number of ITGA5+ macrophages in the infarcted myocardium 7 days after myocardial infarction.Moreover, new immunofluorescence images clearly show the ITGA5+ macrophages, by including the 2 channels and the merged image (Figure1D).Supplemental Figure III shows the complete time course.We have also revised Supplemental Figure XI to show separate channels that clearly demonstrate ITGA5 loss in macrophages of tamoxifen-treated CX3CR1 CreER ;ITGA5fl/fl mice 3)Supplemental Figure XI: Please mention whether or not p values in the table of panel C were adjusted for multiple testing.

Reviewer # 3 (
Remarks to the Author): Review Li et al. " Macrophage-specific alpha5 integrin protects the infarcted heart from adverse remodeling, by stimulating angiogenesis through PI-3K and FAK pathways"

4 )
The experiments in figure6use another Cre-line (CX3CR1) to recapitulate the findings of the knockout using LyzM-Cre.It is unclear why the authors didn't use this model from the start as they want to analyze the role of ITGA5 in macrophages?To what extent are the other myeloid cells affected from ITGA5 loss in the first (myeloid cell) model?What is the (mature) macrophage content within the LyzM positive myeloid cell fraction in the myocardial infarction model?

a)
We have examined the time course of ITGA5 expression in macrophages using flow cytometry.The findings demonstrate that expression of ITGA5 in CD11b+/Ly6G-/CD64+/MerTK+ macrophages peaks 7 days after MI.The new data are shown in Figure 1.The gating strategy for this experiment is shown in Supplemental Figure II.

1 )
new confocal imaging experiments to show the localization of ITGA5 in macrophages.Specifically, the following revisions were implemented: a. ITGA5 localization in CSF1R+ macrophages is now clearly demonstrated in triple fluorescence images (ITGA5/CSF1R(EGFP)/DAPI, 7 days after MI).The new images are shown in Figure 1D-I of the revised version of the manuscript, and in Reviewer Figure 1 in the current response.Reviewer Figure I: Dual immunofluorescence was performed in myocardial sections from infarcted CSF1R EGFP macrophage reporter mice (7 days coronary occlusion), combining ITGA5 staining (red) and CSF1R (GFP) labeling.Panels D-F show low magnification images (scalebar=50m).Panels G-I show high magnification images (Scalebar=30m, panel G shows the rectangular area indicated in panel D. Abundant ITGA5+ macrophages were noted in 7-day infarcts (D-I, arrows).CSF1R-negative cells with vascular, or fibroblast morphology (D-F, yellow arrow) also expressed ITGA5.Quantitative analysis is shown inFigure 1J-K.The complete time course of macrophage ITGA5 expression after MI is shown in Supplemental Figure III.b. the figure showing the time course of ITGA5 expression has been replaced with a new one with better quality images (Supplemental Figure III) c. new confocal images are shown to demonstrate ITGA5 localization in CSF1R+ cells.Confocal images are shown in Figure 1L-N and in this response as Reviewer Figure II.Reviewer Figure II: Confocal microscopy shows that ITGA5 is localized not only on the macrophage surface (white arrows), but also in the cytoplasm (yellow arrow), likely reflecting de novo synthesis of ITGA5, followed bysubsequent shuttling to the cell membrane.Scalebar=10m.Moreover, additional confocal images of ITGA5/CSF1R(EGFP)/WGA/DAPI-stained sections are shown in Supplemental Figure IV.WGA binds to glycoproteins on the cell membrane and extracellular matrix, and was added to provide additional evidence of cell surface localization.Itga5 is a transmembrane protein.However, the Itga5 signal in IF panels (Fig. 2 and Sup Fig. III) appears cytoplasmic and appears to co-localize with the cytoplasmic GFP from the CSF1RGFP reporter.Can authors explain this? 4) Could the authors confirm the presence of Itga5 on the macrophage cell surface?If not, the authors should comment on the apparent cytoplasmic localization of Itga5 in macrophages and explain how cytoplasmic Itga5 regulates signaling to induce VEGFA.Our flow cytometry studies were performed in the absence of permeabilization protocols.Thus, our flow cytometry data (Figure 1) and the confocal imaging studies (Figure 1L-N and Supplemental Figure IV) clearly show that ITGA5 is localized on the macrophage cell surface.The figures in the revised version of the manuscript clearly illustrate the cell membrane localization.
Fig. III (Compare IIIA with IIIE, and IIIF).Arrows in IIIF point to different cells in different panels.For example, the right arrow in the middle panel points to a round green cell.This is not the same cell in the right-most (merged) panel.3) Arrow placements in IF panels are sloppy and should be checked (see additional specific comments below).